US20150209275A1

US20150209275A1 - Fast Dissolving Ocular Insert

Info

Publication number: US20150209275A1
Application number: US14/427,195
Authority: US
Inventors: Yahya Essop Choonara; Lisa Claire Du Toit; Pradeep Kumar; Viness Pillay; Raeesa M. Moosa; Rafeeq Jhetam
Original assignee: University of the Witwatersrand, Johannesburg
Current assignee: University of the Witwatersrand, Johannesburg
Priority date: 2012-09-11
Filing date: 2013-09-11
Publication date: 2015-07-30
Also published as: ZA201502355B; US20150274765A1; EP2895147A2; EP2895153A1; WO2014041485A1; WO2014041489A2; WO2014041489A3; EP2895147B1; CN104797239A; CN104797239B; US9284341B2

Abstract

This invention relates to a pharmaceutical dosage form, particularly to a topical ocular pharmaceutical dosage form comprising a polymeric matrix of polyethylene oxide block copolymer, preferably polyoxyethylene-polyoxypropylene block copolymer and hydroxpropyl cellulose, and a pharmaceutically active ingredient incorporated within the matrix. The invention extends to a method of manufacturing the pharmaceutical dosage form.

Description

FIELD OF INVENTION

BACKGROUND TO THE INVENTION

Ocular disorders, conditions or diseases of the anterior segment of eye require an effective topical, locally acting, drug delivery system that ensures penetration of the drug through the cornea, effective therapeutic drug levels and patient compliance. Topical methods for drug delivery to the anterior eye remain one of the most convenient and common means of drug delivery. In particular, the use of eye drops still dominates as the drug delivery system of choice by patients. Thus, focus has been directed to topical systems such as eye drops and gels which currently are the most popular pharmaceutical dosage forms for topical ocular use available on the market.
Despite the convenience and compliance using eye drops, limitations are evident. Most eye drops remain on the eye for between about 5 to about 25 minutes and only about 1 to about 10% of drug or active pharmaceutical ingredient (API) is absorbed (Anumolu et al., 2009). This results in low bioavailability of ophthalmic drugs when administered using topical dosage forms such as eye drops (du Toit et al., 2011). Patients often have difficulty administering conventional eye drops due to the following reasons: Firstly, compliance is a problem since eye drops typically have to be administered several times during the course of the day. Secondly, certain patients with conditions such as arthritis have difficulty administering the eye drops since handling of the eye drop bottle poses a problem. A certain level of pressure has to be applied to the bottle to expel the eye drop and a dispensing end of the bottle has to be specially directed such that an expelled eye drop engages and/or enters the eye. These patients are then disadvantaged due to lack of control and synchronization of the bottle together with the eye-lid closure reflex.
Over the years, various attempts have been made to optimize the bioavailability of topically administered ocular drugs. Some of these approaches include: viscous vehicles and hydrogels (Rajas et al., 2011), facilitated transport via prodrugs (Järvinen and Järvinen 1996), nanoparticles (Diebold and Colonge, 2010; Zimmer and Kreuter, 1995), contact lens delivery systems (Ali et al., 2007) and penetration enhancers (Kikuchi et al., 2005). These systems have been developed and used to increase corneal contact time and allow for improved corneal drug penetration to the anterior segment of the eye (Weyenberg et al., 2004). However, their use is associated with several disadvantages. For example, gels and ointments can blur vision and cause reflex blinking. Contact lenses may prove difficult to insert and requires removal (Saettone and Salimen, 1995).
Limitations with respect to the anatomical and physiological aspects of the eye itself are also evident. When drug is administered topically to the eye, reflex closure of the eye lid occurs due to a foreign-body sensation (Urtti, 2006). This results in the liquid preparations being forced out or even prevents entry into the anterior eye altogether. Also, the flow of lacrimal fluid removes administered compounds from the surface of the eye and this fluid, which may entrain the administered drug or API, is then drained by the nasolacrimal duct (Barar et al., 2009). The cornea serves as a protective layer of the eye and comprises several layers (Patel et al., 2004). The corneal epithelium is the most anterior layer of the eye and is approximately 0.1 mm in thickness (Mannermaa et al., 2006). On the outside is the multicellular epithelium which consists of 56 layers of cells and these tight junctions and hydrophobic regions pose a barrier to drug penetration (Chang, 2010; Rathore and Nema, 2009). Therefore, the corneal epithelium limits drug absorption to the anterior segment of the eye.
Apart from improving bioavailability of topically administered ocular drugs, an improved administration system is required to ensure patient compliance and that the drug reaches the target site. As mentioned, eye drops may be difficult to administer and are easily flushed out thus resulting in poor bioavailability especially in elderly patients (Davies, 2000). Thus, there has been effort directed in developing novel drug delivery systems for ocular use. Solid drug delivery systems such as mini-tablets have been investigated as potential vehicles to overcome the challenges associated with the use of conventional eye drops. Using the concepts of improving contact time and bioavailability of ocular preparations, mini-tablets for ocular drug delivery have been formulated. Mini-tablets can be defined as tablets with a diameter of about 2 to about 3 mm (Lennartz and Mielck, 1998). Some advantages that these systems offer over conventional eye drops include: avoiding flushing out of the drug by lachrymation and drainage once administered; increased corneal contact time due to the use of bioadhesive polymers and the fact that the outer layers of mini-tablets swell upon hydration and as the hydration front penetrates into the tablet core providing a gradual release of drug or API over time; low cost and ease of manufacture; and such systems have been reported not to induce mucosal irritation (Ceulemans et al., 2001; Weyenberg et al., 2003; Weyenberg, 2005). Disadvantages of these systems are that they dissolve in the range of hours and may be displaced from their original position within the eye after administration causing some degree of irritation.
Rapid disintegrating systems are defined as systems that have rapid disintegration in less than about 1 minute without water or a small amount of water (about 1 to about 2 mL) (Fu et al., 2004). Advantages of rapid disintegrating solid drug delivery systems include: good stability, accurate dosing, ease of manufacture and ease of use (Dobetti, 2001, Chandrasekhar et al., 2009). Common uses of such rapid disintegrating drug delivery systems include use in oral methods of administration for geriatric or paediatric patients. In terms of ocular delivery, the concept of fast disintegrating systems has not been thoroughly explored. The advantages of solid drug delivery systems to ocular surfaces include: comfortable and non-irritant use due to small and light structure; convenient form of administration and accurate dosing compared to liquid formulations (Virely and Yarwood, 1990); rapid hydration of system due to porous structure thus reducing foreign body sensation in the eye (Refai and Tag, 2011); and preservatives are not required as in the case of conventional liquid formulations. Once in contact with the lachrymal fluid on the eye surface, disintegration of the system occurs with rapid release of the pharmaceutically active compound.
Research has been conducted on several rapid disintegrating solid delivery systems for ocular use typically comprising polymeric matrices that gel on insertion into the lower cul-de-sac of the eye for controlled release of pharmaceutically active compound.
Using solid hydrophilic matrices in the form of mini-tablets for ocular use has gained popularity over the years. Further, the use of freeze-dried mini-tablets may provide a quicker disintegration time compared to conventional tablets. The lyophilization or freeze drying method is defined as: sublimation of frozen water in the sample from a solid phase to a gas phase under reduced pressure (Tsinontides et al., 2004). The process involves selection of polymer-excipient concentrations and subjecting the solution to freezing followed by lyophilization of the sample. This results in the formation of the porous product. On exposure to fluid, ingress of fluid into the matrix occurs with resultant dissolution and release of the pharmaceutically active compound.
The concept of employing lyophilized systems in topical ocular drug delivery has been investigated. Freeze drying results in a solid dosage form which further results in reduced degradation reactions. In addition, easier handling during transportation and storage is noted (Virely and Yarwood, 1990). In terms of solid rapidly disintegrating delivery systems, they offer the following advantages: i) comfort of use due to small and light structure, ii) convenient form of administration and accurate dosing compared to liquid and/or gel formulations (Carpenter et al., 1997), iii) benefit of liquid formulation in a solid form, iv) preservatives are not required since water content is below 5% and this does not favour microbial growth (Süverkrüp et al., 2004) and v) rapid hydration of system due to porous structure thus reducing foreign body sensation in the eye (Refai and Tag, 2011).
Ocular tolerability of solid dry drops comprising hydroxypropylmethylcellulose (HPMC) was demonstrated in healthy human eyes in phase 1 studies (Diestelhorst et al., 1998). Lux and co-workers (2003) concluded that a HPMC lyophisilate displayed higher sodium florescein levels in the anterior eye compared to liquid eye drops in human volunteers. A superior intraocular bioavailability of sodium florescein was seen with a 1% hypromellose lyophisilate compared to eye drops and thus a pharmacokinetic advantage was demonstrated (Abduljalil et al., 2008). A study by Refai and Tag (2011), investigated freeze-dried sponge-like ocular mini-tablets for ocular keratitis treatment comprising sodium carboxymethyl cellulose, HPMC, xanthan gum, chitosan and Carbopol 943P. These mini-tablets showed significant sustained release of acyclovir and good bioadhesive properties and permeation across the cornea in rabbits.
However, there exists a need for novel, easy to manufacture, rapid pharmaceutically active ingredient (API) releasing solid pharmaceutical dosage forms for topical ocular use.

Definitions

The following terms used through the course of this patent specification shall have the following meanings:

- Instantly soluble—implies rapid ingress of fluid into a dosage form and immediate onset of dissolution and/or disintegration of the dosage form;
- Biocompatible—not displaying toxic effects on biological tissue; and
- Biodegradable—capable of being decomposed through biological processes.

SUMMARY OF THE INVENTION

In broad terms this invention relates to a pharmaceutical dosage form for the delivery of at least one active pharmaceutical ingredient (API) to a desired target site in a human or animal, the pharmaceutical dosage form comprising at least one matrix forming polymer, preferably two matrix forming polymers, the two matrix forming polymers preferably being from the class of polymers comprising polyethylene oxide block copolymers as well as cellulosic polymers such as hydroxpropyl cellulose (HPC), such that the at least one matrix forming polymer is formed into a solid pharmaceutical dosage form.
According to a first aspect of this invention there is provided a pharmaceutical dosage form for the delivery of at least one active pharmaceutical ingredient (API) to a target site in a human or animal, the pharmaceutical dosage form comprising a homogenous polymeric matrix consisting of a polyethylene oxide block copolymer and hydroxpropyl cellulose (HPC).
In a preferred embodiment of the invention the polyethylene oxide block copolymer may be polyoxyethylene-polyoxypropylene block copolymer. The polyethylene oxide block copolymer may be a Pluronic polymer, preferably Pluronic F-68 (PF-68).
The pharmaceutical dosage form may further comprise an anti-collapsing agent. The anti-collapsing agent may comprise an amino acid chain, preferably the amino acid chain having 1 amino acid residue, more preferably 2 amino acid residues.
In a preferred embodiment of the invention, the anti-collapsing agent comprises an amino acid chain having two amino acid residues, preferably diglycine.
The pharmaceutical dosage form may further comprise a lyoprotectant, preferably maltodextrin.
The pharmaceutical dosage form may further comprise a superabsorbent polymer, preferably polyacrylic acid sodium salt (PAA-Na salt).
The pharmaceutical dosage form may further comprise at least one active pharmaceutical ingredient (API) homogenously dispersed therein selected from the group: prostaglandin analogs such as latanoprost, beta blockers such as timolol, alpha agonists such as brimonidine, carbonic anhydrous inhibitors such as dorzolamide hydrochloride, or combinations of these such as timolol and dorzolamide hydrochloride or timolol and brimonidine.
In a preferred embodiment of the pharmaceutical dosage form, the dosage form comprises:

- a homogenous polymeric matrix consisting of a Pluronic F-68 and hydroxpropyl cellulose (HPC);
- an anti-collapsing agent, preferably diglycine;
- a lyoprotectant, preferably maltodextrin;
- a superabsorbent polymer, preferably polyacrylic acid sodium salt (PAA-Na salt); and
- an active pharmaceutical ingredient (API).

The pharmaceutical dosage form may be formed into a solid ocular pharmaceutical dosage form for the delivery of the at least one active pharmaceutical ingredient (API) to a region of the eye.
In a preferred embodiment of the invention the solid ocular pharmaceutical dosage form is formed as a tablet, particularly a mini-tablet having substantially circular and/or discoid shaped dimensions wherein the thickness is about 2 mm and the diameter is about 3 mm.
According to a second aspect of this invention there is provided a method of manufacturing a pharmaceutical dosage form of the first aspect of this invention, the method comprising the steps of:

- (a). dissolving polyethylene oxide block copolymer and hydroxpropyl cellulose (HPC) in a liquid medium, preferably deionized water to produce Solution 1;
- (b). adding to Solution 1 a lyoprotectant, preferably maltodextrin; a superabsorbent polymer, preferably polyacrylic acid sodium salt (PAA-Na salt); and an anti-collapsing agent, preferably diglycine, to produce Solution 2;
- (c). freezing Solution 2; and
- (d). lyophilizing the frozen Solution 2 to form the solid ocular pharmaceutical dosage form.

Step (b) may further comprise adding an active pharmaceutical ingredient (API) to Solution 1.
Step (c) may typically take place for about 24 hours at about −82° C. and Step (d) may take place at about −42° C. for about 24 to about 48 hours.
The method may include freezing Solution 2 in polyvinyl chloride (PVA) blister packs of predetermined size in order to produce dosage forms having substantially circular and/or discoid dimensions of about 2 mm in thickness and 3 mm in diameter.
The composition of dry components dissolved in the deionized water prior to freezing in % w/v may be as follows: the polyethylene oxide block copolymer, preferably PF-68 may be in the range of about 1 to about 5% w/v; the hydroxpropyl cellulose (HPC) may be about 0.5% w/v; the lyoprotectant, preferably maltodextrin, may be in the range of about 1 to about 5% w/v; the superabsorbent polymer, preferably polyacrylic acid sodium salt (PAA-Na salt), may be about 0.25% w/v; and an anti-collapsing agent, preferably diglycine, may be about 0.25% w/v.

DESCRIPTION OF THE DRAWINGS

The invention is now described by way of example only, with reference to the accompanying diagrammatic drawings, in which

FIG. 1 shows a) a flow diagram showing simplified methodology employed for the fabrication of the pharmaceutical dosage form, b) a schematic of the pharmaceutical dosage form, and c) a digital image of a typical pharmaceutical dosage form in accordance with this invention;

FIG. 2 shows a typical a) force-time profile for matrix resilience calculation, b) force-distance profile for the determination of matrix tolerance, yield value and energy of absorption and c) force-distance profile for computation of Brinell Hardness Number (BHN);

FIG. 3 a calibration curve of the drug or API, Timolol (TM) in simulated aqueous humour (SAH) (pH7.4, 35° C.) at 295 nm N=3 in all cases (SD≦0.0543);

FIG. 4 shows a typical distance-time graph for disintegration profiling;

FIG. 5 shows scanning electron microscopy (SEM) images depicting the porous lyophilized matrix surface of the pharmaceutical dosage form according to the invention (magnification 615×);

FIG. 6 shows stereomicroscopy images for visualization of the rapid dissolution process (magnification 10×) of the pharmaceutical dosage form according to the invention;

FIG. 7 shows a bar graph depicting drug entrapment efficiency (DEE) of design formulations for the pharmaceutical dosage form according to the invention;

FIG. 8 shows graphs indicating release of active pharmaceutical ingredient (API) of a) formulations 1-5, b) formulations 6-10 and c) formulations 11-13;

FIG. 9 shows digital images of: a) incubation process, b) eggs placed in an equatorial position after albumin removal, c) observation of embryo formation on day 3 and d) preparation of egg for sample testing;

FIG. 10 shows digital images outlining the step-wise in vivo studies: a) left eye of New Zealand Albino rabbit, b) insertion of the OISED into the cul-de-sac, c) aqueous humour aspiration, d) removal of surrounding tissue, e) enucleation of the eyeball, f) complete removal of the eye;

FIG. 11 shows a digital image of the OISED in comparison to a US one Dime coin;

FIG. 12 shows FTIR spectra of components of the OISED;

FIG. 13 shows DSC thermograms of a) pure drug timolol (TM) maleate, b) native PF68, c) native maltodextin (MD), d) physical mixture, e) DL final OISED formulation f) ADSC graph drug-loaded OISED formulation, g) TGA graph of pure TM, h) DF OISED formulation and i) DL OISED formulation (N=3 in all cases);

FIG. 14 Porosity analysis plots of drug-loaded optimized OISED formulation: a) linear isotherm, aa) with corresponding SEM images of optimized OISED formulation, b) BET surface area of optimized OISED formulation, c) BJH adsorption cumulative pore volumes of optimized OISED formulation, d) BJH desorption cumulative dA/dD pore areas of optimized OISED formulation, e) typical t-plot generated for optimized OISED formulation (N=3 in all cases);

FIG. 15 shows a graphical representation of drug/API flux of the optimized OISED formulation and TM drug dispersion (error bars indicate standard deviation);

FIG. 16 shows a 2D chromatograph of TM and DS;

FIG. 17 shows graphs depicting a) in vitro OISED drug release results and b) in vivo OISED and commercial eye drop concentrations;

FIG. 18 shows graphs depicting: a) residual plots, b) in vitro release, c) in vivo absorption and d) point-to-point Level AIVIVC model plot; and

FIG. 19 shows digital images depicting corneal layer with a) DL OISED at 30 minutes and b) with DF OISED at 30 minutes, c) DL OISED at 240 minutes and d) DF OISED at 240 minutes.

DETAILED DESCRIPTION OF THE DRAWINGS

In broad terms this invention relates to a pharmaceutical dosage form for the delivery of at least one active pharmaceutical ingredient (API) to a desired target site in a human or animal, the pharmaceutical dosage form comprising at least one matrix forming polymer, preferably two matrix forming polymers, the two matrix forming polymers preferably being from the class of polymers comprising polyethylene oxide block copolymers as well as cellulosic polymers such as hydroxpropyl cellulose (HPC), such that the at least one matrix forming polymer is formed into a solid pharmaceutical dosage form. It is to be understood that the dosage form may be substantially homogenous or may be formed to be layered, either like an onion or like a sandwich. The matrix forming polymers facilitate providing structural integrity to the dosage form.
According to a first aspect of this invention there is provided a pharmaceutical dosage form for the delivery of at least one active pharmaceutical ingredient (API) to a target site in a human or animal, the pharmaceutical dosage form comprising a homogenous or mono-layered polymeric matrix consisting of a polyethylene oxide block copolymer and hydroxpropyl cellulose (HPC). The pharmaceutical dosage form may be a solid ocular pharmaceutical dosage form in the form of a solid eye drop for the delivery of API to a region of the eye. The HPC typically acts as a matrix forming polymer to facilitate providing structural integrity to the solid eye drop.
The solid ocular pharmaceutical dosage form comprises at least one active pharmaceutical ingredient (API) homogenously dispersed therein.
The solid eye drop is light weight and porous in nature. Typically, the solid eye drop is inserted into the eye, preferably administered to the cul-de-sac of the eye. The solid eye drop is soluble upon contact with the mucosal surface of the eye such that dissolution and/or disintegration initiates substantially instantaneously upon contact with the mucosal surface. The rapid dissolution and/or disintegration causes release of API to a region of the eye. Typically, the released API moves through the cornea to posterior regions of the eye.
The particular chemical composition of the solid eye drop causes certain mechanical properties, particularly its porosity facilitates the ingress of liquid media which causes dissolution and/or disintegration and associated API release. The applicant found that the specific combination of a polyethylene oxide block copolymer and HPC, preferably Pluronic F68 and HPC, produced a solid dosage form having a inter-connecting network of pores. These pores facilitated in providing the dosage forms rapid disintegration characteristics in use, and contributed to its rigidity prior to use. The API can be at least one selected from the group, but not limited to: prostaglandin analogs such as latanoprost, beta blockers such as timolol, alpha agonists such as brimonidine, carbonic anhydrous inhibitors such as dorzolamide hydrochloride, or combinations of these such as timolol and dorzolamide hydrochloride or timolol and brimonidine.
The chemical composition of the solid eye drop is biocompatible and biodegradable in nature. Although the preferred embodiment later herein described comprises Pluronic F-68 this should not be seen as limiting. The invention may very well employ the use of other known polyethylene oxide block copolymers in the stead of Pluronic F-68, or other polymers from classes of polymers which the aforementioned can be categorized under.
The solid ocular pharmaceutical dosage form may further comprise an anti-collapsing agent, preferably diglycine; a lyoprotectant, preferably maltodextrin; and a superabsorbent polymer, preferably polyacrylic acid sodium salt (PAA-Na salt). The dosage form is typically formed into a tablet, particularly a mini-tablet, having substantially circular and/or discoid shaped dimensions wherein the thickness is about 2 mm and the diameter is about 3 mm.
It is important to understand that the pharmaceutical dosage form according to this invention may be formulated as an oral wafer matrix, a graft lubricant, a chromatography gel, a wound dressing, a mesh, a degradable bone fixation glue, a degradable ligament glue and sealant, a tendon implant, a dental implant, a reconstituted nerve injectable, a disposable article, a disposable contact lens, an ocular device, a rupture net, a rupture mesh, an instant blood bag additive, an instant haemodialysis additive, an instant peritoneal dialysis additive, an instant plasmapheresis additive, an inhalation drug delivery device, a cardiac assist device, a tissue replacing implant, a drug delivery device, an endotracheal tube lubricant, a drain additive, and a dispersible suspension system.
According to a second aspect of this invention there is provided a method to manufacture the pharmaceutical dosage form of the first aspect of this invention, the method comprising the steps of:

Step (b) typically further includes adding an active pharmaceutical ingredient (API) to Solution 1. The method may include freezing Solution 2 in polyvinyl chloride (PVA) blister packs of predetermined size in order to produce dosage forms having substantially circular and/or discoid dimensions of about 2 mm in thickness and about 3 mm in diameter. The freezing typically takes place for about 24 hours at about −82° C. Lyophilization may take place at about −42° C. for about 24 to about 48 hours.
The method employed in the manufacture of pharmaceutical dosage forms according to this invention was the lyophilization process which will be described below in detail. This method offers the advantages of simplicity, reproducibility, cost-effectiveness and generation of a stable yet rapidly soluble system.
Design of Experiments (DOE) is defined as a mathematical strategy for setting up experiments in such a manner that the information required is obtained as efficiently and precisely as possible (Lewis et al., 1999; Anderson and Whitcomb, 2007). This statistically based methodology was employed in order to obtain formulations for the purpose of characterization. Tests carried out on the manufactured dosage forms included: textural analysis, disintegration profiling, moisture content studies, scanning electron microscopy (SEM), stereomicroscopy, drug entrapment efficiency (DEE) and in vitro drug release studies.
Representative examples of the invention are described in detail hereunder. The representative examples of the dosage form were formulated as solid topical ocular pharmaceutical dosage forms also termed solid eye drops or instantly soluble solid eye drops (ISEDs).

EXAMPLE 1

Manufacturing & Analyses

Materials

Hydroxypropylcellulose (HPC) (M_w=80 000 g/mol) (Klucel®, Hercules Incorporated, Willington, Del., USA), glycyl-glycine (diglycine, DG) (M_w=132.12 g/mol) (Fluka BioChemika, Belgium), Interfix 1001 Polycaprolactone CAPA® 6400 (Interfix CC, Johannesburg, South Africa), poly(acylic acid sodium salt) (PAA-Na salt) (M_w=5100 g/mol), Maltodextrin (MD) (dextrose equivalent 4.0-7.0) and Pluronic® F-68 (M_w=8400 g/mol) all purchased from Sigma-Aldrich (St. Louis, Mo., USA). All other reagents were of analytical grade and used as supplied. The drug or API is Timolol maleate salt (TM) (M_w=432.49 g/mol) purchased from Sigma-Aldrich (St. Louis, Mo., USA).

Development of an Experimental Design

A two-factor, three-level Face Centered Central Composite Design (FCCCD) was applied for the construction of a second order polynomial model describing the effect of formulation constituents on the characteristics of the system. Pharmaceutical dosage forms according to the invention, in the form of instantly soluble solid eye drops ISEDs, of various combinations were prepared in accordance with the FCCCD (Table 1). The term “instantly soluble” was assigned to the dosage form since it dissolved rapidly (see Definitions section).

TABLE 1

Thirteen formulations of pharmaceutical dosage forms according to the invention
generated from the Face-Centered Central Composite Design with variations of MD and PF68.
Each Formulation contained 5 mg (0.5% before lyophilization) of the active pharmaceutical
ingredient (API) in the form of Timolol maleate salt (TM) in order to produce a drug-loaded (DL)
dosage form. All % w/v is prior to lyophilization.

	PF68
	(% w/v)
	(Pluronic	Maltodextrin	HPC	DG (% w/v)	PAA-Na	API (TM)
Formulation	F68)	(MD) (% w/v)	(% w/v)	(diglycine)	(% w/v)	(% w/v)

1	1	5	0.5	0.25	0.25	0.5
2	5	3	0.5	0.25	0.25	0.5
3	3	5	0.5	0.25	0.25	0.5
4	1	3	0.5	0.25	0.25	0.5
5	5	1	0.5	0.25	0.25	0.5
6	3	3	0.5	0.25	0.25	0.5
7	3	3	0.5	0.25	0.25	0.5
8	3	3	0.5	0.25	0.25	0.5
9	5	5	0.5	0.25	0.25	0.5
10	3	3	0.5	0.25	0.25	0.5
11	1	1	0.5	0.25	0.25	0.5
12	3	3	0.5	0.25	0.25	0.5
13	3	1	0.5	0.25	0.25	0.5

Preparation of Polymeric Solutions and Synthesis of Pharmaceutical Dosage Forms According to FCCCD

Aqueous solutions of polymer were prepared in various concentrations in accordance with the FCCCD. Table 1 depicts formulations generated from the design based on influential components as determined from preformulation studies. Components were dissolved in 100 mL deionized water and agitated for 30 minutes until complete dissolution had occurred. Samples of 150 μL were injected into each mould of the polyvinyl chloride (PVC) blister packs employing a 1 mL syringe. Samples were then frozen (Sanyo Ultralow Temperature freezer, MDF-U73V, Sanyo Electric, Japan) for 24 hours at −82° C. to solidify the product. The product was placed in a lyophilizer (Labconco Freeze-Dry Systems, Labconco Corp., Kansas City, Mo., USA) for 48 hours to extract excess water. On attainment of the samples they were stored in glass vials in the presence of 2 g desiccant sachets. FIG. 1 depicts the methodology and schematic of the proposed formulation.
The method above was utilized to prepare a drug-loaded (DL) ISEDs. When manufacturing a drug-free (DF) ISED the API or drug was omitted from the remaining components which were dissolved in the deionized water prior to freezing.
All analytical experiments outlined below as part of Example 1 were conducted on drug-loaded (DL) ISEDs where Timolol maleate salt (TM) was the API.

Determination of the Physicomechanical Properties of the Pharmaceutical Dosage Forms According to the Invention

Textural analysis was used to characterize the compressibility of the dosage form using a Texture Analyzer (TA.XTPlus, Stable Microsystems, UK). The following tests were conducted for characterization at room conditions (about 25° C.). The following textural properties were determined:
1. Matrix Resilience (MR): Resilience can be defined as the ability of a material to return to its original position or state after stress has been applied to it. Resilience of the dosage form provides an elucidation of the ability of the dosage form to withstand an applied stress. An analyzer (TA.XTPlus, Stable Microsystems, UK) was fitted with a suitable 10 mm diameter delcin probe for resilience measurement. Force-time profiles were generated and analyzed.
The MR (%) was determined by finding the ratio between anchors 2 and 3 and between anchors 1 and 2 (FIG. 2 a).
2. Energy of Absorption (EA): The energy of absorption is an indirect indication of the porosity of the dosage form. A highly porous dosage form will exhibit a greater value for the energy of absorption. The energy of absorption is calculated by determining the area under the curve (AUC) of a profile illustrating force (N) and distance (m) as depicted in FIG. 2 b.
3. Matrix Yield Value (MYV): Matrix yield value assists in the determination of the strength of the surface structure of the dosage form. This is determined by obtaining a gradient between anchors 1 and 2 of a force-distance profile (FIG. 2 b).
4. Matrix Tolerance (MT): Matrix tolerance indicates the overall strength of the dosage form. The gradient between anchors 1 and 3 provides the matrix tolerance value. This is the point of total collapse of the dosage form (FIG. 2 b).
5. Brinell Hardness Number (BHN): Hardness is described as the resistance of an object to permanent shape change when a force is applied. Brinell hardness is an indication of the force required to indent the surface of the dosage form. Brinell hardness was assessed using a ball point probe. Force-distance profiles were generated and assessed (FIG. 2 c). The indentation hardness was represented by a conversion to the Brinell Hardness Number (BHN) (N/mm²) using the following equation:
$\begin{matrix} BHN = \frac{2 F}{π D (D - \sqrt{D^{2} - d^{2}})} & Equation 1 \end{matrix}$
Where:
F=force generated from indentation (N) obtained from maximum peak of curve,
D=diameter of ball probe indenter (3.125 mm) and
d=indentation depth which is half the probe diameter (1.5625 mm)
Textural profiling was determined as per the settings in Table 2.

TABLE 2

Parameters employed for textural analysis

Parameters	MR¹	BHN²

Test mode	Compression	Force
Pre test speed	1.0 mm/sec	1.0 mm/sec
Test speed	0.5 mm/sec	0.5 mm/sec
Post test speed	1.0 mm/sec	1.0 mm/sec
Compression force	—	40N
Trigger force	0.05N	0.05N
Load cell
5 kg	5 kg
Compression strain	50%	—
Compressive distance		1.5625 mm

¹Matrix resilience
²Brinell hardness number

Disintegration profiling of the pharmaceutical dosage form according to the invention Disintegration testing of rapidly dissolving dosage forms or delivery systems is an important test to be carried out for the evaluation of the time taken for such a system to dissolve in use. The Texture Analyzer method has been employed for the evaluation of disintegration of fast dissolving oral wafers (Dor et al., 2000; El-Arini and Class, 2002; Fu et al., 2004). Advanced testing utilizing improved methods were conducted employing a Texture Analyzer (TA) (Stable Micro Systems, Surrey, UK) with a flat, cylindrical probe. The probe head was magnetically attached to the shaft which was screwed onto the load cell carrier. This allowed for preparation of 5 samples at a time and quick removal and interchanging of probe heads was done. The dry ISED was attached to the probe head by means of a thin strip of double sided adhesive tape across its diameter. This was lowered into a Perspex test vessel containing 5 mL Simulated Lachrymal Fluid (SLF, pH 7.4, 35° C.) (Table 3). Upon entering the medium and quickly reaching an immersed perforated platform the Texture Analyzer applied a minimal force (0.098N) for a chosen period of time. The clear vessel allowed for observation of the process. Typical distance-time profiles were generated according to set parameters (Table 4) and disintegration rate (mm/sec) and end point disintegration time (s) were determined.

TABLE 3

Preparation of simulated lachrymal fluid (SLF)

	Components	Quantity

NaHCO₃	218	mg
NaCl	678	mg
CaCl₂•2H₂O	8.4	mg
KCl	138	mg
Deionized water
100	mL

(Gonjari et al., 2009)

TABLE 4

Parameters employed for disintegration profiling

	Parameters	Settings

	Test mode	Compression Hold until reset
	Pre test speed	2.0 mm/sec
	Test speed	3.0 mm/sec
	Post test speed	10.0 mm/sec
	Force	0.098N
	Trigger force	0.029N
	Data Acquisition rate	500 points per second
	Maximum tracking speed	5 mm/sec

Surface Morphology by Scanning Electron Microscopy (SEM)

The in-depth surface morphology and internal structure of the dosage form was visualized by the use of scanning electron microscopy (SEM) (FEI Phenom™, Hillsboro, Oreg., USA). Samples were mounted on a spud and gold plated by the sputter-coater (SPI modul™ sputter-coater and control unit, West Chester, Pa. USA).Samples were then viewed under the SEM at different magnifications.

Visualization of Dissolution Process Employing Stereomicroscopy

The visualization of the solubilization of the ISED was determined by microscopy using light illumination for images in a 3-dimensional level. A stereomicroscope (Olympus SZX7 stereomicroscope, Olympus, Japan) connected to a digital camera (CC 12, Olympus, Japan) and image analysis system (AnalySIS® Soft Imaging System, GmbH, Germany) was employed. The unhydrated and hydrated samples were imaged to observe the entrance of fluid into the sample on a microscopic level.

Computation of Drug Entrapment Efficiency (DEE)

DEE was conducted by dissolving the ISEDs in SLF and spectrophometrically analyzing the solution at 295 nm employing a standard calibration curve for timolol maleate (the drug or API in this representative example) (FIG. 3) in Simulated Aqueous Humour (SAH, Ph 7.4, 37° C.) (Table 5) for determination of drug or API content. All tests were conducted in triplicate. The following equation was employed for the DEE calculation:
$\begin{matrix} DEE = \frac{D_{a}}{D_{t}} \times 100 & Equation 2 \end{matrix}$
Where:
DEE=Drug entrapment efficiency,
D_a=the actual quantity of drug (mg) measured by UV spectroscopy and
D_t=the theoretical quantity of drug (mg) added in the formulation.
FIG. 3 shows calibration curves of TM in SAH (pH7.4, 35° C.) at 295 nm (SD≦0.0543).

TABLE 5

Preparation of simulated aqueous humour (SAH)

	Components	Quantity

	NaCl	6400 mg
	KCl	750 mg
	CaCl₂	480 mg
	MgCl
₂	300 mg
	Sodium acetate	3900 mg
	Sodium Citrate	1700 mg
	Deionized water	1000 mL

	(Giannola et al., 2008)

In Vitro Drug Release Studies

The Franz diffusion cell is a method that can be used for studying the diffusion of drug or API from semisolid ocular dosage forms (Gorle and Gattani, 2009; Gilhotr et al., 2010; Gilhotra et al., 2011). A Franz diffusion cell consists of a donor chamber and a receptor chamber with a membrane clamped in between. Commercial cellophane membranes (M_w=12000, Sigma-Aldrich Corp. St. Louis, Mo., USA) were used to simulate the corneal epithelial barrier for ocular penetration of drug. Membranes were presoaked in dissolution media overnight. The diffusion cell donor chamber contained 2 mL SLF which was maintained to simulate tear volume and solid eye drops were placed in the donor compartment in contact with the membrane. The receptor solution of 12 mL Simulated Aqueous Humour (SAH, pH 7.4, 35° C.) (Table 6) was contained in the receptor chamber. The membrane was placed such that the surface was in contact with the receptor solution which was continuously stirred by a magnetic stirrer at 20 rpm to simulate blinking. Aliquots of 2 mL were withdrawn from the receptor compartment at regular intervals (30, 60, 120, 240, 360 minutes after insertion) and replaced an equal volume of dissolution medium. The samples were analyzed spectrophotometrically at 295 nm (Hewlett Packard 8453 Spectrophotometer, Germany) to determine the drug release. Tests were conducted in triplicate. The dissolution data was analyzed by calculation of the Mean Dissolution time (MDT). This is determined as the sum of individual period of time during which a specific fraction of the total drug or API is released (Pillay and Fassihi, 1999; Govender et al., 2005). MDT_50%data point was selected for the design formulations. The following equation was used for the determination of MDT:
$\begin{matrix} MDT = \sum_{i = 1}^{n} t_{i} \frac{M_{t}}{M_{\infty}} & Equation 3 \end{matrix}$
Where:
M_t=the fraction of dose released in time t_t
t_t=(t_i+t_i-1)/2 and
M_∞=loading dose

Results and Discussion

Textural Profile Analysis of Instantly Soluble Solid Eye Drops (ISEDs)

The physical characteristics of the ISEDs as a dosage form are essential for determination of the stability during storage and use of the product in the eye. It provides an indication of the integrity of the formulations. The numerical values obtained from the analysis at room conditions are listed in Table 6. Low MD and PF68 concentrations resulted in ISEDs that were fragile. Higher MD and PF68 concentrations produced well formed and easily removable ISEDs. Higher MD imparted rigidity and improved hardness of the formulations. Formulation 6, 7, 8, 10 and 12 comprised the same formulation combinations. An increase in matrix resilience is attributed to higher strength and this is an important parameter to help understand the characteristics of the dosage form. Resilience of these formulations was noted to be higher than other formulations ranging from 4.698 to 6.519%. Elevated BHN values are due to higher stability characteristics. The BHN values did not vary much among the formulations. Highest BHN value was seen for formulation 1 (2.062 N/mm²). Energy of absorption was used as an indication of indirect quantification of matrix porosity (Patel et al., 2007). Formulation 1 displayed the highest energy of absorption (0.026 J) which indicates a superior stability between the surface and polymeric network and thus a more robust sample. More pores within a dosage form cause a higher energy of absorption, and was observed as such for formulation 1. Highest tolerance was noted for formulation 7 (19.531 N/mm) which indicates a high overall matrix strength. In terms of yield value, the highest value was seen for formulation 1 (2.9180 N/mm). The correlation between yield and tolerance values is such that initially a low yield value is observed and subsequent to fracturing of the dosage form this energy is dissipated resulting in complete collapse.

TABLE 6

Textural analysis results conducted on design formulations

	Matrix		Matrix
	yield	Matrix	absorption	Matrix
	value	tolerance	energy	resilience	BHN
Formulation	(N/mm)	(N/mm)	(J)	(%)	(N/mm²)

1	2.918	17.402	0.026	8.469	2.062
2	2.121	11.057	0.019	4.589	2.042
3	2.874	15.592	0.023	6.519	2.055
4	1.395	15.878	0.020	5.439	2.058
5	1.307	16.935	0.018	4.712	2.061
6	1.035	16.164	0.018	5.274	2.032
7	2.261	19.531	0.018	5.257	2.057
8	1.932	13.814	0.022	4.698	2.059
9	0.947	11.941	0.022	4.137	2.054
10	1.096	14.892	0.018	4.934	2.050
11	1.289	11.513	0.016	3.814	2.055
12	2.171	15.849	0.016	6.519	2.048
13	1.462	12.454	0.022	5.895	2.054

Results are expressed as the mean of at least three measurements, SDs (standard deviation) obtained were within: yield value ≦0.010, tolerance ≦0.059, absorption energy ≦0.002, resilience ≦0.081, BHN≦0.011.

Disintegration Profiling

Disintegration end point times (EPDTs) were determined by noting the end point on graphs. The EPDT was taken as the time taken for complete disintegration to occur as the solid eye drop was immersed in SLF. Instantaneous disintegration was noted for all the formulations due to the porous nature of the ISED and quick ingress of the fluid. All formulations dissolved to form a liquid with the exception of formulations 2, 5 and 9. This is due to the higher PF68 concentration (5%). The polymer displayed a tendency to form a gel-like residue with increasing concentration. This can be attributed to PF68 being a solid hydrophilic block co-polymer which upon heating and increased concentrations displays a tendency to form a low viscosity thermoreversible gel-like substance (Maghraby and Alomrani, 2009). Formulation 11 displayed the fastest EPDT of 0.200 s while formulation 9 had the slowest time of 3.340 s. Disintegration rate was taken as the first gradient of the descending region from the onset of disintegration. The highest disintegration rate was noted for formulation 11 (10 mm/sec). This correlated with the highest disintegration time. This can be explained in terms of the low PF68 and MD concentration which produced a fragile matrix of formulation 11 and thus resulted in rapid breakdown of the matrix. Table 7 indicates values obtained from the study while FIG. 4 shows a typical profile.

TABLE 7

Disintegration results obtained

	End Point
	disintegration	Disintegration
	time	rate
	(EPDT)	(DR)
Formulation	(s)	(mm/sec)

1	0.3200	6.250
2	3.0800	0.649
3	0.4800	4.167
4	0.2700	7.407
5	3.0400	0.658
6	0.5200	3.846
7	0.5800	3.448
8	0.5400	3.704
9	3.3400	0.598
10	0.5600	3.571
11	0.2000	10.00
12	0.5500	3.636
13	0.6300	3.175

Surface Morphology

The lyophilized ISED appeared to have a sponge-like surface and an interconnecting network with the presence of pores (FIG. 5). FIG. 5 shows SEM images depicting the porous lyophilized ISED (magnification 615×). The pores were generally circular or asymmetrical and widespread across the matrix surface. Entrance of aqueous media into these pores allowed for rapid hydration and disintegration of the ISED. It was noted that a relationship between pores and disintegration time existed. Formulations with a spongy pore appearance had a quicker disintegration time. It is the specific combination of compounds comprising the ISED that provide for the forming of pores, and which impart the physico-chemical and/or physico-mechanical properties described herein, particularly the characteristic of rapid dissolution in use.

Stereomicroscopy

Microscopic imaging of the ISED was performed to aid with visualization of the rapid dissolution process (FIG. 6). FIG. 6 shows stereomicroscopy images for visualization of the rapid dissolution process (magnification 10×). This was apparent by the images captured. Initially the unhydrated solid eye drop and water front were distinctly separated. Upon addition of water, rapid ingression was noted and the ISED dissolved in less than 1 second.

Drug Entrapment Efficiency (DEE)

The purpose of calculating the DEE was to determine the percentage of API or drug loaded into the formulations. A homogenous dispersion of API is required. This would ultimately affect the API release profile and a high DEE is desirable with a maximum of 100%. DEE for the dosage form ranged from 79-96%. Table 8 outlines DEE values calculated and FIG. 7 is a graphical representation of DEE. The API in these examples was timolol (TM) as described above.

TABLE 8

Drug entrapment efficiency (DEE) of various formulations

	Formulation	DEE (%)

	1	81.573
	2	83.295
	3	94.451
	4	93.262
	5	88.227
	6	85.987
	7	90.139
	8	95.225
	9	91.293
	10	89.982
	11	78.922
	12	96.132
	13	92.918

	Results are expressed as the mean of three measurements, SD obtained was ≦2.517.

In Vitro Drug Release

Drug release was determined and plots are depicted in FIG. 8. The mean dissolution time (MDT) was calculated in order to determine the drug or API release characteristics of the formulations (Table 9). With respect to drug release patterns, low MDT values represent rapid release and high MDT values indicate a prolonged release. All formulations showed initial burst release. This can be attributed to the porous nature of the system since it was instantaneously soluble upon contact with SLF due to the presence of hydrophilic polymers (PF68 and HPC) and the drug or API contained therein was released. This is favorable for this dosage form since it allowed for rapid dissolution which results in minimal irritation and with the added advantage of reduced loss due to washout as the initial form is a solid. Formulations 2, 5 and 9 which contained a higher concentration of PF68 (5%) showed longer MDT values due to the gel-like properties of the polymer with increased concentration.

TABLE 9

Mean dissolution times (MDT) calculated for drug or
API release characteristics

	Formulation	MDT_50%(minutes)

	1	25.0000
	2	54.5000
	3	42.1000
	4	27.2500
	5	55.0000
	6	49.5000
	7	49.0000
	8	48.7500
	9	56.5000
	10	48.2000
	11	21.2000
	12	48.1000
	13	45.2500

	Results are expressed as the mean of three measurements, SD obtained was within ≦0.430.

CONCLUSIONS

Herein described is a rapidly disintegrating pharmaceutical dosage form formulated for specific topical ocular delivery of an API, and preferably termed an instantly soluble solid eye drop (ISED). The dosage form having sufficient strength. Textural analysis revealed a robust dosage form was manufactured. Disintegration testing demonstrated that instant dissolution was achieved and thus rapid liberation of API or drug was possible. Surface morphology allowed for visualization of the porous surface of the formulations. The pharmaceutical dosage form formulated may be suitable for ocular use due to the biocompatibility of the polymers and excipients employed. Furthermore, the rapid disintegration would allow minimal irritation to the ocular surface due to reduced foreign body sensation. In addition, the method of production was simple and relatively time effective thus possible cost-effectiveness in terms of manufacturing may be advantageous. The applicant found that the specific combination of a polyethylene oxide block copolymer and HPC, preferably Pluronic F68 and HPC, produced a solid dosage form having a inter-connecting network of pores. These pores facilitated in providing the dosage forms rapid disintegration characteristics in use, and contributed to its rigidity prior to use.

References for Example 1

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EXAMPLE 2

Further Testing

The focus of further testing was to gauge the in depth pertinent pharmaceutical properties and in vivo behavior of a pharmaceutical dosage form, particularly a solid ocular pharmaceutical dosage form, namely, an optimized instantly soluble solid eye drop (OISED) in accordance with the invention. In brief, thermal and molecular transition analysis showed congruent findings with no incompatibility between components of the OISED. Porositometric studies confirmed the presence of interconnecting pores across the matrix surface. Drug release kinetic evaluation predicted that best model fit was first-order release (R²=0.98). The HET-CAM test indicated an irritation score of 0 with the inference of good tolerability. Ex vivo permeation across excised rabbit cornea showed an improved steady state drug flux (0.00052 mg.cm^−2.min ⁻¹) and permeability co-efficient (1.7×10⁻⁴cm.min⁻¹) for the OISED device compared to pure drug and a marketed eye drop preparation. Ultra-performance liquid chromatography (UPLC) analysis indicated drug (timolol maleate, TM) and internal standard (diclofenac sodium) elution at 0.5 and 1.5 s respectively. Gamma irradiation served as an effective terminal sterilization procedure. In vivo results revealed a peak concentration was reached at 104.9 minutes. In the case of eye drops a lower C_maxwas obtained (1.97 ug/mL). Level A point-to-point IVIVC plots a Wagner Nelson method indicated a satisfactory R²value of 0.84. The biodegradability and biocompatibility by histological toxicity studies was confirmed.

1. Introduction

Formulation of an ocular pharmaceutical dosage form requires special attention due to the intricacy and sensitive nature of the eye. Solid ocular pharmaceutical dosage forms for use in the eye have been studied due to the advantages noted. These include lack of flushing, more accurate dosing and better stability. One such example is the application of fast dissolving systems, conventionally for oral application, to the eye surface. However, the process of lyophilization may have an impact on the physical properties, thermal behavior and chemical stability of the polymers involved (Guo et al., 2000). Additionally, the porosity of a freeze-dried system is significant since the voids within a matrix often display a relationship with the appearance and in vitro behavior of the dosage form (Sznitowska et al., 2005). As pointed out by Vargas and co-workers (2007), in order to evaluate novel drug delivery systems, the use of animal models is required but has the disadvantage of being costly, difficult and time-consuming. Thus, alternative means have to be delved into. The Hens Egg Choriollointic Membrane Test (HET-CAM) is an alternative to the Draize rabbit eye test for the assessment of potential ocular irritants and has been applied in various ocular studies (Gorle and Gattani, 2005; Gilhotra and Mishra, 2012). This test has been accepted in Europe and documented in the current EU guidance to The UN Globally Harmonized System of Classification and Labeling of Chemicals (UN GHS) (EU, 2009; Scheel, 2011).
Topical ocular drug delivery using a conventional system such as liquid eye drops is associated with several drawbacks. The following specific points are of paramount importance when considering the use of such formulations: i) a very small volume of lachrymal fluid (7-9 μL) is found on the eye surface while a dropper bottle dispenses more than this present volume of liquid. Majority of this is then flushed out upon instillation which consequently results in a loss of contained drug; ii) instillation of the liquid, due to foreign body sensation, may trigger a flow of tears which forces more of the liquid to be lost and iii) content of substances for preservation of the solution which may not be well tolerated with triggering of blinking and further loss of drug.[1-3] Furthermore, these dosage forms are convenient and widely available, but have poor bioavailability due to dilution in lachrymal fluid upon eye drop instillation and subsequent removal by the naso-lacrimal drainage system.[4] Thus, continual administration of liquid drops is needed in order to achieve therapeutic effects. This can result in poor patient compliance and in turn hinders the overall treatment process.
In order to improve the bioavailability of topical ocular drugs, several other systems have been proposed and investigated. These include formulations with an increased viscosity and thus increased corneal contact time such as inserts and viscous polymers.[5,6] However, these systems may suffer from the disadvantage of transient blurring of vision and foreign body sensation, which in turn may negatively affect patients willingness to use such systems.
Drug delivery systems research is making strides as investigators gain better insight to the factors that affect their mechanism of action. Rapid disintegrating systems (also called ‘fast-melt’ or ‘fast-dissolving’) as novel replacements for conventional solid tablets have been investigated. These systems are solid dosage forms which disintegrate rapidly, usually in the time frame of minutes or seconds. Most commonly applied for oral use, these systems quickly dissolve once exposed to salivary fluid and this minimizes the need for external water or fluid medium.[7-9] To help improve the administration, compliance and bioavailability of topically administered drugs the concept of fast disintegrating systems can be implemented to ocular drug delivery systems.
Acquiring in vivo data from any pharmaceutical dosage form in the animal model is essential to corroborate the feasibility of the novel system in human patients. Prior to that, it is important to ensure that the dosage form is rendered sterile before proceeding to in vivo analysis. This is also a requirement of Good Manufacturing Practice (GMP) for pharmaceuticals. Of note in the selection of a method is that the procedure should ideally be validated and not impinge on the quality and properties of the product intended for sterilization. For purposes of this further testing, gamma (γ)-irradiation was employed. Commonly, this method has been used for ocular systems such as mini-tablets, implants and gels (Petrich and Rosen 1995, Weyenberg et al., 2006; Choonara, 2010). The purpose of selecting this method was due to the apparent advantages such as: safety, simplicity, controlled conditions and importantly, no residual activity after the process. The use of this method is recognized by the American National Standards Institute (ANSI), the American Association of Medical Instrumentation (AAMI) and the International Standards Organization (ISO) (Martin, 2012).
In terms of data analysis, pharmacokinetics studies the relationship of the active pharmaceutical ingredient (API) or drug in the body. It allows for a mathematical means to interpret data obtained from in vivo analysis. However, from an ocular perspective, this is the study of absorption, distribution and elimination of drug after topical instillation on the eye surface (Mayers et al., 1991). In vitro in vivo correlation (IVIVC) is a concept can had been described by the Food and Drug administration FDA as model that shows the relationship between the in vitro drug dissolution and in vivo absorption results. In vivo absorption can be determined by means of model independent Wagner Nelson method (one compartment model) or Loo-Riegelmin (multi-compartment model). There are mainly 4 levels of IVIVC i.e. A, B, C and multiple C. These levels are based on the ability to reflect the complete plasma time profile of which Level A provides a point-point to point relationship and is often preferred. IVIVC serves to support a dissolution method and assists in quality control during manufacturing (Leeson, 1995). To determine the pK parameters complicated calculations have to be carried out, however software packages can be used for time effective results. Some programs such as WinNonlin may be expensive and have a steep learning curve, thus alternatives are required. One such example is the Microsoft Excel (MSE) 2003 add in PKSolver. This is a freely available add-in programme that has demonstrated to be simple to install and operate, compatible with MSE 2007 and 2010, has various calculation options and most importantly values for parameters generated have been compared to that of WinNonlin with acceptable results (Zhang, 2010).
Therefore, the aim of this further testing was to evaluate the OISED in New Zealand Albino rabbit eye. A novel UPLC method for the detection of timolol maleate (TM) in aqueous humour was developed and the pharmacokinetic parameters of in vivo release were determined in comparison to a marketed eye drop preparation in the New Zealand Albino rabbit eye. Finally, the compatibility of the OISED with the ocular tissue via histology was assessed.

2. Materials and Methods

2.1 Materials

Hydroxypropylcellulose (HPC) (M_w=80 000 g/mol) (Klucel®, Hercules Incorporated, Wilingtion, Del., USA), glycyl-glycine (diglycine, DG) (M_w=132.12 g/mol) (Fluka BioChemika, Belgium), poly(acylic acid sodium salt) (PAA-Na salt) (M_w=5100 g/mol), Maltodextrin (MD) (dextrose equivalent 16.5-19.5), Pluronic® F-68 (PF68) (M_w=8400 g/mol), Sodium Dodecyl Sulphate (SDS) (M_w=288.38 g/mol) and Timolol maleate salt (TM) (an example of an active pharmaceutical ingredient (API) or drug) (M_w=432.49 g/mol) were all purchased from Sigma-Aldrich (St. Louis, Mo., USA), diclofenac sodium (another example of an API or drug) (M_w=318.13 g/mol) were purchased from Sigma Aldrich, ammonium acetate (M_w=77.09 g/mol, Saarchem, Muldersdrift, South Africa), glacial acetic acid (Associate Chemical Enterprises, Southdale, Johannesburg), acetonitrile 200, perchloric acid and methanol (UPLC grade, ROMIL™, Johannesburg), Glaucosan® 0.5% eye drops (Hexal Pharma (SA)(PTY)(LTD) and double deionized water was obtained by use of a Millipore filter (Millipore water purification system, Millipore, Molsheim, France).

2.2 Methods

2.2.1 Attainment of an Optimized Formulation of the ISED

A two-factor, three-level Face Centred Central Composite Design (FCCCD) as shown in Example 1 (Table 1) was applied for the construction of a second order polynomial model describing the effect of formulation constituents on the characteristics of the system. ISEDs of various combinations were prepared in accordance with the FCCCD. In order to determine the optimal formulation, several responses were tested: Textural characteristics, disintegration testing and in vitro drug release. Constraint optimization predicted an optimized formulation of MD and PF68 concentrations with sufficient strength and rapid disintegration as carried out in previous studies using Minitab®.

2.2.2 Preparation of Polymeric Solutions and Synthesis of the Optimized ISED (OISED)

Aqueous solutions of polymer were prepared in accordance with the optimized formulation components obtained using: HPC 1% w/v, PAANa 0.25% w/v, DG 0.25% w/v, MD 5% w/v, and PF68 2.94% w/v. (The optimized formulation comprised 0.5% timolol maleate as the API). Components were dissolved in 100 mL deionized water and agitated for 30 minutes until complete dissolution had occurred. Samples of 150 μL were injected into each mould of the polyvinyl chloride (PVC) blister packs. Samples were then frozen (Sanyo Ultralow Temperature freezer, MDF-U73V, Sanyo Electric company, Japan) for 24 hours at −82° C. to solidify the product. The product was placed in a lyophilizer (Labconco Freeze-Dry Systems, Labconco Corp., Kansas City, Mo., USA) for 48 hours to extract excess water. Lyophilized drug-loaded OISED samples were stored in glass vials in the presence of 2 g desiccant sachets.
The method above was utilized to prepare a drug-loaded (DL) OISED. When manufacturing a drug-free (DF) OISED the API or drug is omitted from the remaining components which are dissolved in the deionized water prior to freezing.
All analytical experiments were conducted on the drug-loaded (DL) OISED wherein TM is the API.

2.2.3 Determination of the Molecular Vibrational Transitions

Fourier transform infrared (FTIR) spectroscopy is used for the detection of interactions between native polymers and blends as well as to detect whether a specific functionality is present. FTIR was carried out to detect vibration characteristics of chemical functional groups in response to infrared light interactions. A Perkin Elmer Spectrum 2000 FTIR spectrometer with a MIRTGS detector, (PerkinElmer Spectrum 100, Llantrisant, Wales, UK) was used. Samples were be prepared as pellets against a blank ZnCn pellet background at a wave number ranging from 4000-650 cm⁻¹and a resolution of 4. The spectrum software (Spectrum 100) was used for interpretation of the results.

2.2.4 Determination of Thermophysical Properties by Temperature Modulated Differential Scanning Calorimetry (TMDSC)

Thermal analysis was carried out to determine the thermal properties of the formulations as per the FCCD shown in Table 1. A Temperature Modulated Differential Scanning Calorimeter (TMDSC) (Mettler Toledo, DSC1, STAR^eSystem, Swchwerzenback, ZH, Switzerland) equipped with software for computation evaluation of numerical values was used. The glass transition temperature (Tg), melting temperature (Tm) and temperature of crystallization (Tc) were determined. Temperature calibration was attained by a melting transition of 6.7 mg indium. Samples (10 mg) were weighed out on aluminium crucibles, sealed and tested within a temperature gradient of 0-300° C. under constant N₂purge to reduce the oxidation rate of 1° C./min.

2.2.5 Determination of Supplementary Thermal Properties via Thermal Gravimetric Analysis (TGA)

Supplementary thermal characterization was conducted using a thermogravimetric analyser (TGA) (PerkinElmer, TG-IR 8000, Llantrisant, Wales, UK) in order to determine weight variations of samples as a function of temperature. The instrument was purged with N₂to prevent the occurrence of any undesirable reactions. Samples were placed in a pan using tweezers followed by heating between 50-550° C. at a rate of 10° C./min. Weight vs. temperature plots were generated and analysed using Pyris™ software.

2.2.6 Complementary Surface Morphology and Porositometric Analysis

The in-depth surface morphology and internal structure of the matrix was visualized. This was done by the use of scanning electron microscopy (FEI Phenom™, Hillsboro, Oreg., USA). Samples were mounted on a spud and gold plated by the sputter-coater (SPI module™ sputter-coater and control unit, West Chester, Pa. USA). Samples were then viewed by the SEM at different magnifications. In addition, the visualization of the surface of the OISED was determined by microscopy using light illumination for images on a 3-dimensional level. A stereomicroscope (Olympus SZX7 stereomicroscope, Olympus, Japan) connected to a digital camera (CC 12, Olympus, Japan) and image analysis system (AnalySIS® Soft Imaging System, GmbH, Germany) was employed. The porosity of the OISED was determined by employing a porositometer (Micromeritics ASAP 2020, GA) with the use of Brunauer-Emmett-Teller (BET) isotherm of adsorption/desorption of nitrogen. The process involved: i) degassing of samples was carried out which involves an evacuation and heating phase, parameters of which are outlined in Table 10. Samples were inserted into tubes and underwent this phase and ii) asdorbtive properties were then determined. Data analysis by means of the following were obtained: i) BET calculation which was obtained from determination of the monolayer volume of adsorbed gas from isotherm results, ii) t-plot calculation for analysis of area and total volume due to micropores in the matrix and iii) Barrett-Joiner-Halenda (BJH) for the determination of the mesopore volume/area distribution which accounts for both the change in adsorbate layer thickness and the liquid condensed in the matrix pore cores.

TABLE 10

Parameters employed for degassing phase of OISEDs

	Parameters	Settings

a) Degassing conditions (evacuation)

Temperature ramp rate	10°	C./min
Target temperature
30°	C.
Evacuation rate	50.0	mmHg/s
Unrestricted evacuation from	30.0	mmHg
Vacuum set-point	500	mmHg
Evacuation time	60	min

b) Degassing conditions (heating)

Ramp rate	10°	C./min
Hold temperature
30°	C.
Hold time	900	min
Hold pressure
100	mmHg

	c) Analysis conditions (adsorptive)
	Adsorptive	Nitrogen gas (N₂)

Maximum manifold pressure

925

kPa

	Nonideality factor	0.0000620
	Density conversion factor	0.0015468

Hard sphere diameter	3.860	A°
Molecular cross-sectional area	0.162	nm²

2.2.8 Ocular Toxicity Evaluation Employing Modified Hen Egg Chorioallontoic (HET-CAM) Test

2.2.8.1 Assay Preparation and Procedure

The methodology involved using hen's eggs (50-60 g fertile) that were purchased from a local hatchery (Bruce Hatcheries, South Africa) and less than a week old. The eggs were placed in commercial incubators (Surehatch, Brakenfell, South Africa) and rotated every 12 hours. On day 3, a hole was made and the albumin was removed. The eggs were sealed by sterile heated parafilm and left in an equatorial position. On day 5 and everyday thereafter eggs were candled for viability and non-viable ones were discarded. On day 10, a window (2×2 cm) was made and 2-3 mL saline (0.9% NaCl) was added and eggs were returned to the incubator. The following samples were prepared for testing: Standard solution: 1% sodium dodecyl sulphate (SDS), test sample: test sample drug-loaded (DL) OISED dissolved in 0.9% NaCl, Placebo sample: drug-free (DF) OISED dissolved in 0.9% NaCl, Control: 0.9% NaCl. The eggs were then dosed with 0.3 mL of samples and observed for 5 minutes for any effects of injury in accordance with Table 11 and this was noted. Saline was used as a control for comparison purposes. Eggs were scored for severity of any reaction and time taken to occur (Table 11). At the end of the assay the embryos were discarded immediately by placing the eggs into a freezer −80° C. The following features were used for observation of injury: hemorrhage, vascular lysis and coagulation. FIG. 9 depicts the procedure for the test.

TABLE 11

Scoring chart employed for the HET-CAM test (10)

		IS
Effects	Score	ranges	Inference

No visible haemorrhage	0	0-0.9	Non-irritant
Visible membrane discoloration	1	1-4.9	Mildy-irritant
Structures covered due to partial	2	5-8.9	Moderately
haemorrhage/discolouration			irritant
Structures covered fully due to	3	9-21	Severely irritant
haemorrhage/discolouration

IS = irritation score

The period taken for any untoward reaction to occur was noted and the irritation threshold was determined (highest concentration of samples required for a minimal reaction to occur). The irritation score was calculated by means of Equation 4.
$\begin{matrix} IS = \frac{301 - secH .5 +}{300} \frac{301 - secL .7 +}{300} \frac{301 - secC .9}{300} & Equation 4 \end{matrix}$
Where:
IS=irritation score
Sec=onset of reaction in seconds
H=hemorrhage
L=lyses
C=coagulation

2.2.9 Treatment of Dissolution Data by Comparison of Release Profiles of the OISED and a Commercial Product

A model independent approach was used for mathematical analysis of dissolution data. According to the US FDA, the similarity and difference factors are significant to identify for this purpose. Two factors were determined with the time point set at 360 minutes. The difference factor (f1) is a measurement of percentage error of two curves at each time point. Curves are similar if f1 is close to 0. This was calculated by Equation 5.
$\begin{matrix} f_{1} = \frac{\sum_{j = 1}^{n} \langle R_{j} - T_{j} \rangle}{\sum_{j = 1}^{n} R_{j}} j & Equation 5 \end{matrix}$
The similarity factor (f2) is the logarithmic transformation of the sum-squared error of differences between test and reference products over all time points. Curves are considered similar when f2 is between 50 -100. Equation 6 was employed to determine f2:
$\begin{matrix} f_{2} = 50 \times \log {{[1 + (\frac{1}{n}) \sum_{j = 1}^{n} {\langle R_{j} - T_{j} \rangle}^{2}]}^{- 0.5} \times 100} & Equation 6 \end{matrix}$
Where:
n=sampling number
R and T=reference and test products at each time point j

2.2.10 Ex Vivo Permeation Studies of the Optimized Formulation (OISED) in Comparison to a Commercial Anti-Glaucoma Product

Franz diffusion cell apparatus was used to conduct ex vivo permeation studies. Excised rabbit corneas (11.83±0.04 mm in diameter and area 3.0±0.01 mm²) were mounted on the Franz diffusion cell apparatus (Permagear, Arnie Systems, USA) connected to a heating bath system for temperature control. The diffusion cell donor chamber was filled with 2 mL Simulated tear (lacrimal) fluid (SLF) (Gonjari et al., 2009) (which was maintained to simulate tear volume). 12 mL Simulated Aqueous Humor (SAH, pH 7.4, 37° C.) (Giannola et al., 2007) solution was contained in the receptor chamber. The cornea was placed such that the surface was in contact with the receptor solution which was continuously stirred by a magnetic stirrer at 20 rpm to simulate blinking. Samples of one optimized formulation (OISED), pure drug dispersion and one drop of marketed product (0.5% timolol maleate) were tested in triplicate for comparison purposes. Aliquots of sample were withdrawn from the receptor compartment at regular intervals (30, 60, 120, 180, 240 minutes and 24 hours after insertion) and replaced with an equal volume of dissolution medium. Samples were then subjected to the quantification of drug through spectrophotometric analysis at 295 nm (Hewlett Packard 8453 Spectrophotometer, Germany) to determine the drug release and flux (rate of drug permeation per unit area). Drug flux values were calculated at the steady state per unit area by regression analysis of permeation. Drug flux values were calculated employing Equation 7.
$\begin{matrix} J_{s} = \frac{Q_{r}}{A \times t} & Equation 7 \end{matrix}$
Where:
J_s=drug flux (mg cm⁻²min⁻¹)
Q_r=amount of drug that passed through to the receptor compartment (mg)
A=Active cross-sectional area for diffusion (cm²) and
t=time of exposure (min)
The permeability co-efficient can be described as the velocity of drug diffusion through the cornea based on its mathematical unit using Equation 8.
$\begin{matrix} kp = \frac{J_{s}}{C_{d}} & Equation 8 \end{matrix}$
Where:
Kp=permeability co-efficient (cm.min⁻¹)
J_s=Flux at steady state (mg cm⁻²min⁻¹)
C_d=drug concentration in donor compartment (mg.cm⁻³)

2.2.11 Ultra Performance Liquid Chromatography (UPLC) Methodology

The equipment consisted of an ultra-performance liquid chromatography system (Acquity™ Ultra Performance LC, UPLC, Waters, coupled with PDA detector and ELC detector). Data acquisition and interpretation was conducted using Waters Empower software. Analysis was performed with ammonium acetate (AA): acetonitrile (ACN) (49:51 v/v) as a mobile phase which was pumped at flow rate of 0.25 ml/min in isocratic mode employing a C₁₈column (1.7 μm; 1×100 mm) at 25° C. The wavelength was kept at 295 nm for detection of TM and 2 ul of sample was injected for analysis. The run time was set at 3 minutes. AA buffer was prepared by dissolving 7.7 g in 100 ml water as solvent A. pH was adjusted to 4.5 with glacial acetic acid in a drop-wise manner. ACN 100% was employed as solvent B. TM and internal standard (IS) were prepared and solutions were kept in amber glass vials until use. Standard solutions of drug and IS were prepared and calibration curves were constructed. A mixture in a 1:1 ratio of analyte and IS were prepared. Samples were filtered (0.22 um) and injected into 2 mL vials for analysis.

2.2.12 Aqueous Humor Collection and Drug Extraction Via Liquid-Liquid Procedure

Enucleated rabbit eye balls were collected and punctured for blank aqueous humour sample aspiration (200 uL). Samples were stored in plastic eppendorf tubes at −80° C. until used for analysis. Samples were thawed out (200 uL) and spiked with standard solutions of analytes. An equal volume of IS was added and samples were vortexed for 2 minutes. An equal volume of 6% v/v perchloric acid and methanol were added for deproteination. Samples were centrifuged for 10 min at 1500 rpm (MSE minor laboratory centrifuge, Scientific instruments, West Palm Beach, Fla., USA) and the clear supernatant was removed. This was made up to a volume of 2 mL with mobile phase. Samples were prepared and analyzed as explained above and a calibration curve was constructed (FIG. 2). The lowest standard concentration of the calibration curve was regarded as the lowest limit of quantification (LOQ). Calibration curve obtained for TM in blank aqueous humour samples was constructed. R²of 0.99 was obtained.

2.2.13 In Vivo Characterization of the OISED in the Rabbit Model

Ethics clearance was obtained from the Animal Ethics Screening Committee (AESC) of the University of the Witwatersrand. All animals and biological tissue were handled according to Standard Operating Procedures (SOP) of the Central Animal Services (CAS). Furthermore, guidelines of Association for Research in Vision and Ophthalmology (ARVO) Resolution on the Use of Animals in Ophthalmic Research and Vision Research (Rockville, Md., USA) were followed. Cage activity by means of observation for 1 hour periods daily were used to assess state of well-being. Assessment of wellbeing was determined with the use of a score sheet. Score sheets can be used for routine monitoring and thus contribute to the welfare of the animals. Samples were prepared and packaged into labeled glass poly-tops sealed with a plastic lid. Samples were transported to Isotron (Pty) Ltd. (Isando, South Africa) and irradiated at a dose of 25 KGy (sufficient for sterilization without biological validation according to the European Guideline, 1992). A total of 66 New Zealand Albino rabbits of weight approximately 2.25 kg were used. An initial pilot study was conducted on (6 rabbits). The remaining 60 rabbits were randomly assigned to 2 groups with 30 rabbits each for group 1 and 2. Rabbits were divided into the respective groups (test/placebo group 1 and comparison group 2). A pilot study was conducted on 6 rabbits (1 rabbit for each sampling point) in order to collect samples after OISED insertion. The following groups were assigned: Pilot study (6 rabbits): To determine ease of administration at the defined site and presence of any untoward reactions in the rabbit eye following visual assessment after insertion. Test and Placebo group 1 (30 rabbits): Rabbits in this group received a drug-loaded OISED containing in the cul-de-sac of the left eye. Drug-free OISEDs were inserted into the right eye for the placebo effect. Comparison group 2 (30 rabbits): Rabbits in this group received eye drops of a commercial product (Glaucosan®, 0.5%) in the cul-de-sac of the left eye. Rabbits were anaesthetized with an intramuscular combination of ketamine hydrochloride (40 mg/kg) and xylazine (10 mg/kg) for ocular delivery of the drug-loaded OISED in the left cul-de-sac at time 0. Placebo OISED was inserted into the opposing eye (right). Rabbit eyes were assessed using thorough observation on a macroscopic level. This involved observing any possible effects of the OISED on the eye with reference to Table 13. Evaluation of irritation was conducted according to a scoring system of 0 (absence) to 3 (highest). The untreated eye served as a control. Overall irritation was calculated by addition of the total clinical evaluation scores (A, B, C) (Mishra and Gilholtra, 2008). Samples were withdrawn at 30, 60, 120, 240, 360 minutes and 24 hours after insertion. At each sampling point each animal was euthanised with an overdose of IV sodium pentobarbitone (>50 mg/kg, ˜2 mL) with consequent enucleation (surgical removal of the eyeball). The aqueous humour approximately 200 ul) was aspirated (paracentesis/tapping) employing an insulin syringe fitted with a 26 gauge needle inserted parallel to the iris (Lee and Robinson, 1979). The aqueous humor samples was frozen immediately and stored at —80° C. FIG. 11 provides digital images of the process.

2.2.14 Pharmacokinetic Data Analysis

A one-compartmental pharmacokinetic model (PK) was used for the assessment of ocular results (Mishma, 1981; Lee et al., 1991). This proposes that the drug distributes to a central compartment (aqueous humor) initially followed by a peripheral compartment. PKSolver (Microsoft Excel add-ins) was used for analysis.
The following pertinent pK parameters were determined:
1. Apparent absorption rate constant (k_a)
2. Apparent elimination rate constant (k_e)
3. Peak TM concentration (C_max)
4. Area under the concentration vs. time curve (AUC)
5. Area under the zero and first moment curves from the last sampling time to infinity (AUC_t-∞)
6. Area under the moment curve (AUMC)
7. Peak time (t_max)
8. Mean residence time (MRT)
The following diagnostic criteria were used to determine suitability of the selected model:
1. Co-efficient (R)
2. Weighted sum squares of residuals (SS)
3. Standard error of residuals (SE)
4. Akaike's information criteria (AIC)
5. Schwarz criteria (SC)

2.2.15 In Vitro In Vivo Correlation (IVIVC)

A mathematical model for the relationship between an in vitro property and in vivo response was developed using R-console (version 2.15.3, R foundation for statistical computing) with ivivc package (0.1.6). Input data was the drug release data from the optimized OISED as well as in vivo pK data obtained from UPLC analysis. Level A correlation was selected for IVIVC.

2.2.16 Histological Evaluation of OISED Ocular-Compatibility

Each eye was cut in a sagittal section and each select block of the eye was placed in a histological cassette and processed in an automated tissue processor according to procedures. Following automated tissue processing, wax blocks were prepared and sections of 6 μm were cut on a microtome. The prepared sections were stained in an automated Haematoxylin and Eosin stainer. After staining, the specimen was mounted and the slides examined.

3. Results and Discussion

3.1 Preparation and Sterilization of the OISED

The optimized OISED was prepared as outlined previously. Drug loaded (DL) and drug-free (DF) OISEDs were prepared. FIG. 11 depicts the size of the OISED (diameter 3 mm, thickness 2 mm) in comparison to a US one dime coin (diameter 17.91 mm, thickness 1.35 mm).

3.2 Molecular Vibration Transitions

FTIR spectroscopy is employed for the detection of interaction of polymers or drugs as well as observation of important groups present in constituents. The vibrational bands highlight changes on a molecular level. FTIR spectra and corresponding structures of the pure drug TM, individual maltodextrin (MD) and Pluronic-F68 (PF68), a physical mixture (PM shown on the FIG. 12) and the drug-loaded final OISED formulation (FF shown on FIG. 12) was analyzed for possible interactions (FIG. 12). From the spectrum of pure drug the major peaks were observed thus confirming that the drug was timolol maleate (TM). A quaternary ammonium peak (N—H) was found at 3280.98 cm⁻¹. Presence of an OH group was seen at 3037.96 cm⁻¹. Also, C—O—C stretching (1702.51 cm⁻¹) and C═C aromatic ring (1583.32 cm⁻¹and 1448.66 cm⁻¹) were noted. Furthermore, CH stretching and CH bending were observed at 1352.7 cm⁻¹and 2964.98 cm⁻¹respectively. The broad peak at 2879.95 cm⁻¹was attributed to the OH moeity present in PF68. Additionally, C—O stretching occurred at 1145.98 cm⁻¹. For MD, OH stretching occurred around 3304.07 cm⁻¹. C—O stretching of MD corresponds to peaks at 1059.52 cm⁻¹and 961.79 cm⁻¹. All major peaks of pure drug appeared in the physical mixture indicating that no interaction occurred (3294.94 cm⁻¹, 1586.96 cm⁻¹). For the final formulation, drug peaks were not clearly defined and this could be attributed to the homogenous dispersion within the matrix. In terms of the final formulation, the major components were PF68 and MD. Since no major shifts in peaks were observed, it can be proposed that the absence of any appreciable variations or chemical interaction between polymers or drug was noted and the components maintained their initial structure. The peaks occurring in the region of >3000 cm⁻¹could be attributed to the summation of peaks associated with polymer HPC (3423.75 cm⁻¹), excipients diglycine (DG) (3283.1 cm⁻¹) and sodium polyacrylate (PAANa) (3285.27 cm⁻¹). Hence, this assisted in confirming that the formulation remained stable during the preparation procedure.

3.3 Thermal Analysis of OISED

Thermal analysis of OISEDs is of key importance in order to gather the significant characteristics of the sample. Specifically, the ‘collapse temperature’ which is the temperature over-which the sample displays loss of its structure and collapses during the lyophilization process (Pikal, 1990). This is critical in terms of manufacturing and storage. Testing provides information regarding the possibility of drug-polymer interactions. Analysis was conducted via DSC and thermal curves of the pure drug TM and a drug-loaded final formulation were investigated. In terms of the pure drug a single defined endothermic peak was observed at 203.2° C. confirming its crystalline nature (FIG. 13 a). This corresponded to the melting peak (T_m) of the drug which is the transition from a solid to liquid state with subsequent degradation. In terms of the drug-loaded OISED formulation, PF68 and MD were the major components. Native PF68 produced a distinguishable single step endothermic degradation at 53.9° C. (FIG. 13 b). For pure MD, peaks that occur before 220° C. (106.51° C.) (FIG. 13 c) were possibly due to the elution of water (Elnaggar et al., 2010). The T_mof MD was observed as a broad peak at 230.98° C. In terms of the physical mixture, a less defined broad TM peak in the region of 193.89° C. was seen confirming the presence of the drug (FIG. 13 d). TM uniformly dispersed within the final optimized OISED formulation as well as increased polymer and excipient concentration compared to drug, possibly contributed to the diminished broad T_mof the drug (FIG. 13 e). Furthermore, Simon and co-workers (Simon et al., 2003), reported that a lower endothermic peak of lyophilized samples when compared to the individual components may be due to a decrease in thermal conductivity. This may be as a result of the presence voids/pores within the matrix. Additionally, the endothermic peak was reduced in the final formulation, postulated due to MD (16.5-19.5 dextrose equivalence). It has been investigated to be a lyoprotectant (lyophilization stabilizer) by being amorphous and displaying a diluting ability during the lyophilization process (Corveleyn and Remon, 1996).

3.4 Thermal Gravimetric Analysis

TGA was utilized in order to assist in interpretation of thermal properties of the samples using heat and stoichiometric ratios in determination of the percent by mass of solute. Pure TM displayed a definate weight loss in the region comprising an extrapolated onset of 200° C. and an endset of 350° C. which can be explained as the decomposition temperature (FIG. 13 g). A sharp endothermic peak is observed in this area which is in agreement with the findings of Joshi and co-workers (2009). The derivative curve of TM shows weight loss corresponding to a three step pattern at 231.14° C., 260° C. and 313.95° C. respectively. In terms of the optimized OISED formulations, similar weight loss patterns were noticed. For both DF and DL OISED formulations, sharp weight loss occurred in the region of 200° C.-450° C. (FIG. 13 h and i). Additionally, in the derivative curves for both formulations, 3 points of degradation were noted at approximately 230-240° C., 305-310° C. and 411-413° C. It is explicable that incorporation of drug did not alter the thermal properties.

3.5 Porositometric and Surface Imaging Analysis Comparison

Porosity can be described as the measurement of the spaces within a sample network. With regards to lyophilized matrices, the resultant product is often porous due to the sublimation of water post-freezing. This can be quantified in terms of porosity analysis and visualization by SEM. The basic principle behind this method is the adsorption of nitrogen gas on a material's surface or on the pores, if present. A degassing linear isotherm and the Brunauer-Emmet-Teller (BET) surface area plots are depicted in FIGS. 14 a and b respectively. The linear plot indicates that degassing occurred since the adsorption and desorption isotherms are noted. The presence of a hysteresis loop was noted since a difference in the adsorption and desorption curves were seen as per definition of the loop. The loop infers nitrogen evaporation from pores (Guirguis and Moselhey, 2012). A type 3 hysteresis loop (H3) can be possibly classified due to the isotherms sloping. This implies the presence of slit-like pores. This sample can thus be described as mesoporous (pore diameter between 2-50 A) and macroporous (pore diameter >50 A) according to the IUPAC definition (Condon, 2000).
Corresponding SEM and stereomicroscope images for qualitative analysis enumerated the findings of porosity analysis and allowed for visualization of the surface and pore distribution. Pores were demarcated, interconnecting and circular to assymetrical over the matrix surface. BET plots indicated that a positive value was obtained (27.2052 m²/g, R²=0.99). Explanation of the BET concept is of the assumption that there is a uniform surface exposure and that nitrogen is more attracted to the surface then other nitrogen molecules. BET-C ranges between 5-100. Values <5 implied that the gas-gas affinity was competing with the gas-solid affinity as a result of the significantly reduced surface area and minimization of pores (Everett, 1972; Siminiceanu et al., 2008). A value of 4.785274 was obtained for the formulation tested. A method for determination of pore volume or surface area as a function of pore diameter was proposed by Barret, Joyner and Halender (Barret et al., 1951) BJH adsorption plots, BJH Desorption dV/dlog(D) Pore volume plots with implementation of the Halsey-Faas correction and are seen in FIGS. 14 c and d (Halsey, 1948). The average pore size for BJH adsorption and desorption were 46.644 Å and 53.949 Å respectively. The large pore distribution can be explained on account of the entrapped air spaces within the matrix as a result of the freeze-drying methodology for production. FIG. 14 e displays t-plots based on Lippins, Linsen and de Boer Lippins et al., 1964). This is used for the determination of micropore or mesopore presence <20 Å. Positive BET values enabled the computation of the t-plot. The micropore volume is obtained by extrapolation of the isotherm. A t-plot micropore volume of −0.010393 cm³/g was obtained indicating the presence of micropores. Hence, these findings emphasized that the lyophilization process significantly contributed to the porous nature of the samples and ultimately results in a light structure that rapidly disintegrates. This explains the possible method of disintegration and drug release i.e via fluid filling into the pores of the matrix with subsequent dissolution. Table 12 computes results obtained for optimized drug-loaded samples.

TABLE 12

Results obtained from porosity analysis

	Parameter	DLO

	BET total surface area (m²/g)	27.2052
	BET-C	4.785274
	Qm (cm³/g STP)	6.2495
	BET slope(g/cm³STP)	0.126575 ± 0.007612
	BET y-intercept (g/cm³STP)	0.033439 ± 0.001053
	R²(BET plot)	0.99
	Micropore volume (cm³/g)	−0.010393
	External surface area (m²/g)	38.5234
	R²(t-plot)	0.99
	t-plot slope (cm³/g · Å STP)	2.490519 ± 0.099199
	t-plot y-intercept (cm³/g STP)	−6.718757 ± 0.403672
	Pore diameter range (Å)	7-3000
	Pore thickness range (Å)	3.5-5
	BJH adsorption pore size (Å)	46.644
	BJH desorption pore size (Å)	53.949

	DLO = drug-loaded optimized
	BET-C = intensity of the N₂-surface interaction
	Qm = monolayer surface capacity

3.7 Ocular Toxicity Evaluation

The purpose of employing this test was due to the CAM being tissue that is vascularized (arteries, veins and capillary plexus). The occurrence of any injury with inflammation due to a test substance is an indication of the damage that can occur and be compared to that of the eye surface in vivo and served as the rationale for this test (Spielman, 1991). The HET-CAM test acts as an alternative to the use of mammalian tissue and application of the highly controversial and harmful Draize-irritancy test performed on live animals. In addition, the HET-CAM test is rapid and cost-effective for the evaluation of novel drug-delivery systems. Testing was carried out according to Globally Harmonized System of classification and labeling of chemicals (GHS) guidelines for testing of chemicals (UN, 2009). Results indicated that after exposing the CAM to samples (300 uL) for 5 minutes, drug-free (DF) and drug-loaded (DL) samples of OISEDs were practically non-irritant and therefore well-tolerated with a mean score of 0. This can be attributed to the constituents of the formulation. They are ocular-compatible, biodegradable polymers (HPC and PF68) showed no adverse effects. Furthermore, the drug itself TM, is a standard treatment for glaucoma and its presence displayed no untoward reaction to the CAM. Saline, employed as a control also displayed a score of 0 implying no irritation potential as expected. SDS used as a positive control, showed a score of 2.5 inferring considerable toxicity with disruption of the CAM. It is an organic compound and non-ionic surfactant known to cause cell lysis and is often used for comparison purposes in studies. Equation 1 was used to determine the IS of formulations (0-21). An IS of 0 for NaCl, drug-free (DF) and drug-loaded (DL) formulations were obtained while in contrast, 10.4 for SDS. Notably, NaCl and optimized formulations were virtually non-irritant even at higher concentrations while SLS displayed toxicity at low concentrations. Table 13 outlines results obtained. The CAM remains unaffected when tested with the optimized formulation while disruption is clear with SDS. Thus, this study revealed that both drug-loaded and drug-free solid eye drops were non-irritant to the membrane and can be considered safe for use on the eye surface. It is a pre-requisite for ocular systems to have no adverse reaction on the eye, thus the selected polymers and excipients were suitable for the intended application.

TABLE 13

Results obtained for the HET-CAM test

	Substance	Score	Inference	IS

Negative control
NaCl
0	Non-irritant	0
Positive control
SDS	2.5	Severe	10.40
Samples
DF
0	Non-irritant	0
DL	0	Non-irritant	0

Results expressed as a mean of 3 values
NaCl = saline, DF = Drug free, DL = drug loaded, SLS = sodium lauryl sulphate

3.8 Comparison of Dissolution Data

Both drug-free (DF) and drug-loaded (DL) OISED formulations, since immediate release dosage forms, displayed initial burst release. However, the OISEDs displayed a superior result compared to the commercially available eye drops. The level of similarity was evaluated by means of the similarity and difference factors. A f1 value of 13.69 and a f2 of 61.61 were obtained. A f1 of 0-15 indicates that there are minor differences between the samples tested and f2 between 50-100 indicates the sameness of the samples. This can be used as a surrogate before conducting in vivo studies.

3.9 Determination of Ex Vivo Permeation of OISED in Comparison to a Marketed Product

Permeation capabilities of the optimized formulation compared to the pure drug dispersion of TM and marketed eye drops were tested across excised rabbit cornea samples. Results had a positive outcome indicating that an improved steady state flux was noted for the optimized OISED. (0.00052 mg.cm⁻².min⁻¹) compared to pure drug 2 (0.000378 mg.cm⁻².min⁻¹) and eye drops 3 (0.00039 mg.cm⁻².min⁻¹) (FIG. 15). This can be attributed to the polymers employed in the formulation. PF68 forms a slight gel-like substance due increased temperature of SLF which assists in increases the corneal contact time. Also, HPC is a non-ionic surface-active viscosity adjusting polymer that favours pre-corneal retention thus influencing the drug permeation. A low concentration of the polymers was incorporated to promote rapid disintegration of the device, avoidance of a highly viscous gel-like residue yet allowance of sufficient corneal contact. The influx of fluid in the pores influenced the matrix disentanglement flowed by rapid dissolution and drug liberation. Pure drug solution is diluted and remains in the SLF or on the corneal surface as it is dispersed within a liquid (saline) and thus drug takes longer period to diffuse across the corneal epithelium as opposed to a polymeric device that favours adhesion and corneal interaction. The kp calculated for the samples differed, and this disparity is likely to be due to the difference in composition. As an oral drug, timolol has been classified as a class 1 drug according to Biopharmaceutics Classification system (BCS) (Kasim et al., 2004; Dahan et al., 2009). This infers that it is a highly permeable and soluble drug across the intestinal tissue. On the eye surface, across the corneal epithelium, it also displays favorable permeability due to the advantageous hydrophilic and lipophilic property. The polymers and excipients employed in the optimized OISED formulation are all hydrophilic thus rapid drug liberation and permeation was attained and thus this was suitable for immediate drug release. Table 14 shows differences in kp values.

TABLE 14

Results obtained for permeability co-efficients

	Sample	Kp (cm · min⁻¹)

	Optimized formulation	1.7 × 10⁻⁴
	Pure drug dispersion	1.2 × 10⁻⁴
	Marketed eye drops	1.3 × 10⁻⁴

	Results expressed as a mean of 3 values

3.12 In Vivo Results from the OISED in Comparison to Marketed Eye Drop Preparation

FIG. 16 depicts the chromatograph of TM and IS DS obtained from UPLC 1 hour after analysis. TM was eluted at 0.594 minutes and DS at 1.595 minutes. Intra- and inter-day precision and accuracy and drug recovery were seen as satisfactory (R²=0.98). The developed method used perchloric acid and methanol in order to de-proteinate samples. The mobile phase maintained at a pH of 4.5 was acceptable to allow for drug retention and peak separation. No difference in retention times of TM and DS in blank samples was noted.
During in vivo studies, observations were made to determine the effect of the OISED on the ocular surface following administration. No negative effects were noticed and this served to confirm the results from the HET-CAM test. Table 15 provides the results obtained from scoring.

TABLE 15

Score for clinical assessment of OISED on ocular tissue

Effect	Score DL	Score DF

Conjunctival rednesss
	0	0
Conjunctival chemosis	0	0
Discharge	0	0

Results, as confirmed in previous studies, indicated that peak TM concentrations were achieved around 60-100 minutes in the aqueous humour (Phillips et al., 1985). For the OISED a peak concentration was reached at around 104.9 minutes. In the case of the eye drops a lower C_maxwas obtained (1.97 ug/mL). As expected, the optimized OISED displayed a better drug release profile compared to the eye drops (Table 16). Diagnostic criteria for ‘goodness of fit’ of the selected model revealed that low AIC and SC values showed a good model fit. Reasons for the improved OISED behaviour can be attributed to the following: the eye surface contains hydrophilic glycoproteins termed ocular mucins. As explained by Mantelli and Argueso (2008), these mucins have the function of stabilizing the tear layer to postpone the break-up of this layer. Mucins are found on the cornea as well as the cul-de-sac and polymers have the tendency to bind non-covalently to these mucins. This allows for localization of drug to a specific area and reduces the possibility of drainage as in the case of the drug within a liquid vehicle. HPC is a surface-active polymer that assisted in alteration of the elimination of the drug instilled by means of the OISED. Similarly, the inclusion of PF68 allowed for pre-corneal interaction when exposed to the increased eye temperature (Wahg et al., 2008). FIG. 17 a-b displays the in vivo and in vitro drug release curves for the OISED as well as the eye drops.

TABLE 16

PK parameters and diagnostic criteria for “goodness of fit”
of TM in the aqueous humour following topical application

	Parameters	ISED	Eye drops

Pk parameters
K_a(min⁻¹)	0.012	0.011
K_e(min⁻¹)	0.013	0.01
T_max(min)	100.09	92.2
C_max(ug/mL)	3.14	1.97
AUC_0-t(ng/ml)	628.39	447.03
AUC_t-∞	664.77	496.78
AUMC (ug/ml/min⁻²)	103272.52	91924.99
MRT(min)	155.34	185.04
Diagnostic criteria
R obs pred	0.92	0.86
SS	0.94	1.50
SC	6.17	6.86
SE	1.007	0.866
AIC	7.74	8.032

3.13 In Vitro In Vivo Correlation (IVIVC)

FIG. 18 (a-d) provides a graphical representation of the data obtained. A Wagner Nelson method was employed to calculate the in vivo drug release. Level A point-to-point IVIVC plots indicated a R²value of 0.84. This serves to imply that the in vitro dissolution data can be compared to and may serve as a surrogate to that of in vivo pK data.

3.14 Histological Assessment

Histological analysis was performed to assess the safety of the OISED in the eye surface. The corneal topographical findings from the drug-loaded (DL) and placebo (DF) specimens of the OISEDs from the eyes of the rabbits were recorded. The presence of heterophils as well as the heterophil exocytosis into the conjunctival epithelium appeared minimal. Heterophils are usually indicative of sub-acute or chronic inflammation (Gervais et al., 2011). Absence of defects or inflammation was noticed. Based on the findings, there was minimal pathology or any marked changes and irritation due to the insertion of a drug-loaded device as well as from the placebo administration. FIG. 19 provides images of the corneal layer at different time points.

4. Conclusions

This study aimed to provide an extensive pharmaceutical analysis of an optimized instantly soluble solid eye drop device (OISED). Molecular transition studies and thermal analysis revealed that no incompatibility between drug, polymers and excipients existed. Porosity and morphological examination confirmed the porous-nature of the formulation. First-order release was determined to be the predominant release mechanism as indicated by kinetic analysis. Ocular toxicity studies via the HET-CAM test proved to be efficient for evaluation of samples and determined that the device was non-noxious and thus considered safe for topical ophthalmic application. Comparison with a pure drug dispersion and marketed eye drops for trans-corneal permeation revealed that the optimized formulation had an improved drug flux and permeability co-efficient attributed to the rapid disintegration and presence of hydrophilic adhesive polymers. The application of UPLC for drug detection allowed for an advantageous development of a novel and sensitive method. In vivo assessment indicated that the ISED had improved drug levels in the aqueous humor in comparison to the eye drops. The inclusion of polymers in the OISED allowed for improved corneal adherence and absence of drug drainage as in the case of liquid eye drops. Level A IVIVC correlation indicated an R²value of 0.84. Histological assessment revealed the safety of the device as seen from observational studies. Thus, results from this study showed that the ISED was concluded to display advantageous behavior and considered safe for the eye surface. The applicant found that the specific combination of a polyethylene oxide block copolymer and HPC, preferably Pluronic F68 and HPC, produced a solid dosage form having a inter-connecting network of pores. These pores facilitated in providing the dosage forms rapid disintegration characteristics in use, and contributed to its rigidity prior to use. The specific combination of components comprising the OISED provided for a non-irritant solid ocular pharmaceutical dosage form in use.

Ethical Approval

Ethics clearance was obtained from the Animal Ethics Screening Committee (AESC) of the University of the Witwatersrand. Ethics clearance number: Dec. 5, 2012.
While the invention has been described in detail with respect to specific embodiments and/or examples thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing may readily conceive of alterations to, variations of and equivalents to these embodiments. Accordingly, the scope of the present invention should be assessed as that of the claims and any equivalents thereto, which claims appended hereto.

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Claims

1-15. (canceled)

16. A pharmaceutical dosage form for the delivery of at least one active pharmaceutical ingredient (API) to a target site in a human or animal, the pharmaceutical dosage form comprising a homogenous polymeric matrix consisting of a polyethylene oxide block copolymer, hydroxpropyl cellulose (HPC), and an anti-collapsing agent.

17. The pharmaceutical dosage form according to claim 16, wherein the polyethylene oxide block copolymer is a polyoxyethylene-polyoxypropylene block copolymer, preferably a pluronic polymer, further preferably Pluronic F-68 (PF-68).

18. The pharmaceutical dosage form according to claim 16, wherein the anti-collapsing agent is an amino acid chain.

19. The pharmaceutical dosage form according to claim 18, wherein the amino acid chain has 1 amino acid residue.

20. The pharmaceutical dosage form according to claim 18, wherein the amino acid chain has 2 amino acid residues.

21. The pharmaceutical dosage form according to claim 18, wherein the anti-collapsing agent is diglycine.

22. The pharmaceutical dosage form according to claim 16, further comprising a lyoprotectant.

23. The pharmaceutical dosage form according to claim 22, wherein the lyoprotectant is maltodextrin.

24. The pharmaceutical dosage form according to claim 16, further comprising a superabsorbent polymer.

25. The pharmaceutical dosage form according to claim 24, wherein the superabsorbent is polyacrylic acid sodium salt (PAA-Na salt).

26. The pharmaceutical dosage form according to claim 16, further comprising at least one active pharmaceutical ingredient (API) homogenously dispersed therein selected from the group: prostaglandin analogs, beta blockers, alpha agonists and carbonic anhydrous inhibitors, or combinations of thereof.

27. A pharmaceutical dosage form for the delivery of at least one active pharmaceutical ingredient (API) to a target site in a human or animal, the pharmaceutical dosage form comprising:

a homogenous polymeric matrix consisting of a Pluronic F-68 and hydroxpropyl cellulose (HPC);

an anti-collapsing agent in the form of diglycine;

a lyoprotectant in the form of maltodextrin; and

a superabsorbent polymer in the form of polyacrylic acid sodium salt (PAA-Na salt).

28. The pharmaceutical dosage form according to claim 27, further comprising an active pharmaceutical ingredient (API).

29. The pharmaceutical dosage form according to claim 16, wherein the pharmaceutical dosage form is formed into a solid ocular pharmaceutical dosage form for the delivery of the at least one active pharmaceutical ingredient (API) to a region of the eye.

30. The pharmaceutical dosage form according to claim 29, wherein the solid ocular pharmaceutical dosage form is formed as a tablet.

31. The pharmaceutical dosage form according to claim 30, wherein the tablet is a mini-tablet having circular and/or discoid shaped dimensions wherein the thickness is 2 mm and the diameter is 3 mm.

32. A method of manufacturing a pharmaceutical dosage form comprising the steps of:

(a). dissolving polyethylene oxide block copolymer and hydroxpropyl cellulose (HPC) in a liquid medium, preferably deionized water to produce Solution 1;

(b). adding to Solution 1 a lyoprotectant, preferably maltodextrin; a superabsorbent polymer, preferably polyacrylic acid sodium salt (PAA-Na salt); and an anti-collapsing agent, preferably diglycine, to produce Solution 2;

(c). freezing Solution 2; and

(d). lyophilizing the frozen Solution 2 to form the solid ocular pharmaceutical dosage form.

33. The method according to claim 32, wherein Step (b) further comprises adding an active pharmaceutical ingredient (API) to Solution 1.

34. The method according to claim 32, wherein Step (c) takes place for 24 hours at −82° C.

35. The method according to claim 32, wherein Step (d) takes place at −42° C. for between 24 to 48 hours.

36. The method according to claim 32, wherein Step (c) takes place in polyvinyl chloride (PVA) blister packs of predetermined size in order to produce dosage forms having circular and/or discoid dimensions of about 2 mm in thickness and 3 mm in diameter.